WO2015151128A1 - Method for rapid detection of infection from caga positive helicobacter pylori and diagnostic kit therefor - Google Patents
Method for rapid detection of infection from caga positive helicobacter pylori and diagnostic kit therefor Download PDFInfo
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- WO2015151128A1 WO2015151128A1 PCT/IT2015/000101 IT2015000101W WO2015151128A1 WO 2015151128 A1 WO2015151128 A1 WO 2015151128A1 IT 2015000101 W IT2015000101 W IT 2015000101W WO 2015151128 A1 WO2015151128 A1 WO 2015151128A1
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- pylori
- caga
- suspension
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Definitions
- the present invention concerns a method for the rapid detection of infection from CagA-positive Helicobacter pylori, and a diagnostic kit for carrying out said method. More specifically, the invention concerns an in vitro method for detecting, in samples of gastric mucus, stools or biopsy specimens, the presence of highly pathogenic CagA strains of H. pylori, which method provides results immediately available on performing the test, as well as the diagnostic kits for use in carrying out said method. Background of the invention
- H. pylori is a helix-shaped Gram-negative mi- croaerophilic microorganism that can inhabit the stomach and the duodenum, adapting itself to the inhospitable environment of the gastric mucosa in a quite particular way, to the point that it often fails to produce any detectable symp- toms.
- H. pylori it is considered that more than 80% of the individuals infected with H. pylori are asymptomatic.
- Such microorganism infects more than half of the world population at the level of the gastro-duodenal tract. The transmission route of the infection has not been clarified, although infection seems to be typically acquired in the childhood.
- the stomach is protected from its own gastric juice, made of concentrated hydrochloric acid and rich of digestive enzymes, by a thick layer of mucus which covers the gastric mucosa, within which the Helicobacter may penetrate and survive, resisting to any acid that may possibly reach it thanks to an enzyme produced by such bacterium, urease.
- Urease converts the urea contained in large amounts in the stomach into bicarbonate and ammonium, therefore a basic environment is established in the vicinity of the bacterial colony, such environment being capable of neutralizing the hydrochloric acid of gastric secretions, thereby protecting H. pylori.
- H. pylori Another protective mechanism available to Helicobacter pylori consists in that the natural immune defences of the body cannot reach the microorganism in the gastric mucus.
- the immune system reacts to H. pylori infec- tion by sending leukocytes, T killer lymphocytes and other defence factors, but these cannot reach the infection site, as they cannot easily penetrate through the mucus layer, and remain and accumulate in the site, leaving there their destructive content (consisting of superoxide radicals) when they die. Additional nutrients are sent on the site to assist the white blood cells, and this fact does no more than further feeding Helicobacter itself.
- gastritis develops in a few days, and then it may sometimes progress up to peptic ulcer.
- the causative agent of the damage to the gastric mucosa is not H. pylori itself, but the inflammation and, consequently, the immune response to the microorganism.
- H. pylori is the main cause of chronic gastritis and the most relevant factor for the development of peptic ulcer (Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983; 1 :1273-5).
- H. pylori induces a persistent inflammation (chron- ic gastritis) which may last years without developing with clinical symptoms.
- chronic gastritis may result in the development of gastric or duodenal ulcers.
- gastric cancer gastric adenocarcinoma and non-Hodgkin gastric lymphoma
- H. pylori Moss SF. The carcinogenic ef- feet of H. pylori on the gastric epithelial cell. J Physiol Pharmacol 1999; 50:847-56.
- H. pylori results in a six-fold increase of the risk of gastric cancer. It is believed that chronic gastritis may lead to intestinal metaplasia, which in turn, may degenerate into malignant cancer.
- H. pylori In addition to the mentioned gastroduodenal diseases, infection with H. pylori can aggravate or contribute to the development of coronary heart disease (ischemic heart disease up to myocardial infarction), metabolic disorders (such as increased LDL cholesterol and HDL cholesterol reduction, insulin resistance, etc.), autoimmune diseases (such as autoimmune thyroiditis, systemic sclerosis, etc.) and several other conditions such as osteoporosis, reduced male and female fertility, etc.. For all these reasons, it is necessary to detect early infection and treat it appropriately.
- coronary heart disease ischemic heart disease up to myocardial infarction
- metabolic disorders such as increased LDL cholesterol and HDL cholesterol reduction, insulin resistance, etc.
- autoimmune diseases such as autoimmune thyroiditis, systemic sclerosis, etc.
- several other conditions such as osteoporosis, reduced male and female fertility, etc.
- H. pylori infection is commonly done through the research of bacterial urease in gastric biopsies, during endoscopy, or by looking for bacterial antigens in stool samples. Gastrointestinal endoscopic examination is certainly more reliable because it allows direct viewing of esophageal and gastric mucosa, removal of material by biopsy and culture of the material removed for H. pylori. However, it is an invasive and rather expensive test.
- anti-Hp antibodies in blood would represent a cheap and fairly rapid method of diagnosis, advantageous due to the fact that the detection of specific IgGs in serum has good sensitivity and good specificity.
- its utility is severely limited, as it does not distinguish between active in- fection and past infection.
- the anti-helicobacter antibodies can circulate in the bloodstream for more than two years after recovery from the infection.
- H. pylori HpSA
- the search for fecal antigen of H. pylori, HpSA may identify the presence of antigen in stool and, since this is a sign of an active infection, this test is more reliable than detection of antibodies in the blood. However, with this type of survey the level of false negatives is remarkable, therefore the test is safe only when it is positive.
- a known method of rapid diagnosis based on detection of the fecal H. pylori antigen employs an analytical technique known as coagglutination test, in its variant known as "latex agglutination", which uses polymeric latex particles.
- the original method of coagglutination (Kronvall, G. 1973. A rapid slide agglutination method for typing pneumococci by means of specific antibody absorbed to protein A-containing staphylococci. J. Med. Microbiol. 6: 187-190) is based on a Staphylococcus strain, the strain of Staphylococcus aureus Cowan 1 , which is rich in protein A, a substance having the characteristic of adsorbing in a specific, but non-immunological, manner, immunoglobulins G (IgGs) of subclasses 2 and 4, linking the Fc fragment and leaving the Fab por- tion free to interact with a variety of antigenic molecules.
- IgGs immunoglobulins G
- Staphylococci coated with antibodies thus become a reagent which agglutinates in the presence of the homologous antigen. Agglutination of this S. at/ret/s-protein A complex can be used to detect the presence of antigens (bacterial, viral and parasitic antigens), in body fluids, and for the immunological identification of various pathogens.
- antigens bacterial, viral and parasitic antigens
- staphylococci are replaced by polymer microspheres of uniform size (typically with a diameter of 8 ⁇ ) obtained by emulsion polymerization of hydrocarbon monomers.
- the microspheres known in the field with the name of "latex" particles, exhibit, on their surface, the hydrophilic heads of the hydrocarbon chains that constitute them. These are coated with antisera of bacterial, viral or other origin in a manner similar to that described for staphylococci suspensions, and then the latex sensitization process is similar to that previously described for staphylococci containing Protein A.
- the rapid diagnostic method mentioned above (Blanco et al., "Evaluation of a latex agglutination test (PYLOGEN) for the detection of Helicobacter pylori in stool specimens", Diagn Microbiol Infect Dis. 2009; 63: 349-353) is based on putting in contact a suspension of latex particles sensitized with a monoclonal antibody directed specifically against H. pylori antigens with a stool sample of the subject to be analyzed, and visually checking the possible agglutination, which would be indicative of the presence of H. pylori in the sample.
- H. pylori antigens used for sensitization of the latex particles in the test mentioned above are of polysaccharide nature. According to current knowledge, it is presumed that the different H. pylori strains will not be able to differentiate between fully virulent (because associated with ulcers and cancer) and less virulent on the basis of the constitution of their polysaccharide.
- the main indication of Pylogen is the verification of the eradication treatment, because its sensitivity (75%) in the post-treated patient is slightly better than that obtained in the pre-treatment diagnosis (73%), although the specificity compared to UBT (Urea Breath Test, described below) is less good (93% vs. 100%).
- the examination for H. pylori infection currently regarded as the "gold standard” is the breath test with 13 C-labeled urea (Urea Breath Test), which is still performed in a few centers, mainly because of its cost.
- the examination must be performed after an overnight fast, and involves the oral assumption of sodium citrate, followed, after ten minutes, by an examination of the air exhaled by the patient.
- the patient is then administered one tablet of radio- labelled urea, and a new sample of exhaled air is collected after about 30 minutes. Since, as noted, H. pylori splits urea into ammonia and bicarbonate, the amount of marked CO 2 exhaled after half an hour from a subject with on- going infection will be higher than that exhaled from a healthy subject.
- the cag-positive strains are able to inject an oncoprotein (encoded by one of the cag genes) into colonized gastric cells, named CagA, which increases the risk of developing neoplastic lesions (Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, et al. cag, a pathogenicity island of Helicobacter pylori, encodes type l-specific and disease-associated virulence factors. Proc Natl Acad Sci USA 1996; 93:14648-53).
- H, pylori is a microorganism that has evolved with man. It is part of the genus Helicobacter with thirty different spe- cies that have found their habitat in the stomach of many mammals and in intestines of many vertebrates, mammals and not (e.g. birds). The presence of H. pylori had to confer a selective advantage to individuals hosting it, otherwise this organism would have been lost in the evolutionary path. It is believed that hundreds of thousands of years ago, H.
- pylori was infected by a phage which inserted in its chromosome a DNA fragment taken from a bacterial species that no longer exists (because the nucleotide sequence of this segment is not found in any of the microorganisms examined so far).
- the insertion contains 30 genes involved in virulence, and is called "pathogenicity island” (PAI).
- PAIs were already known in various bacterial species infecting humans and plants, such as Yersinia enterocolitica subsp. pestis, Bordetella pertussis, Agrobacterium tumefaciens: the genes which make up the various PAIs show a very high homology with each other, and all encode for the production of a species of conjugation factor, through which CagA, a protein con- sidered to be an immune-dominant determinant, it is translated, almost injected, into the eukaryotic cell colonized.
- CagA conjugation factor
- the CagA protein is phosphorylated in its tyrosine residues and transformed into an oncoprotein, able to alter the cell morphology and thus disturb the "signaling" systems of the host in such a way that can lead, ultimately, to the development of a gas- trie carcinoma.
- the carcinogenicity of CagA is demonstrated by the observation that transgenic mice expressing CagA systemically develop gastric carcinoma or tumors of the hematopoietic system.
- the bacterium In addition to the CagA protein, the bacterium also transfers part of its peptidoglycan, a constituent of its wall which has the ability to stimulate a strong inflammatory response mediated by transduction of the signal to which NF- ⁇ participates. Inflammatory damage is added to the damage caused by CagA (which also has a strong pro-inflammatory power), and this combined action explains why patients with stomach carcinoma are, or were, all, with very few exceptions, infected with strains of H. pylori expressing CagA.
- transaminases are higher (but still within the normal range) in patients who host a strain expressing CagA. 4. Patients with duodenal ulcers have higher concentrations of monocytes and polymorphonucleates if they have anti-CagA antibodies.
- the frequency of infection by CagA-positive strains is higher in patients with osteoporosis than in controls, and anti-CagA antibody titers corre- late in an almost linear fashion with the risk of pathological fractures in these patients.
- the serologic method is, at the present time, the only method able to evidence an infection by the most virulent strains, since it allows to detect the presence of anti-CagA antibodies in the blood.
- these antibodies can continue to circulate in the blood for as long as 10 years before going back to levels below the detection threshold of the com- mon commercial tests, it is evident that a diagnosis of H. pylori infection allowing to detect high risk strains when the infection is in place is not possible through this method.
- the number and concentration of antigens required to produce a hyperimmune serum are both important factors, as it is also demonstrated by the observation that the latex sensitization with purified IgGs obtained from rabbits immunized with total protein antigens of H. pylori gives rise to a quick and highly visible agglutination when the amount of IgGs used is adequate, but if the amount of IgGs is just halved the quantity of IgGs sensitizing aggluti- nation is considerably less evident.
- CagA is only one of the proteins encoded by the genes of the pathogenicity island cag (which contains about 30 "open reading frames"), each of which encodes for a peptide provided, as CagA, with a high immunogenicity.
- cag is composed by two sections, right and left with respect to an insertion known as IS605, which divides it into two parts without interrupting it. Its presence causes instability of cag, resulting into a possible deletion of an arm of the island. These deletions reduce the virulence of the strains; it happens for some point muta- tions of genes located in the left or right branch, such as cagE and virBIO.
- a rapid diagnostic method for the detection of H. pylori strains expressing the cag pathogenicity island genes, particularly CagA-positive H. pylori strains which method exploits the method of coagglutination with S. aureus or the method of latex agglutination, in which staphylococci or latex particles sensitized with an- ti-CagA antiserum, or with an antiserum anti-total proteins of CagA-positive H. pylori from which other antibodies against H.
- pylori antigens are contacted, in suitable conditions, with a biological sample to be analyzed, and the agglutination or not of the resulting suspension is detected, according to whether or not CagA antigens, and other high virulence proteins of the cag pathogenic island, are present in the sample
- the proposed method according to the invention can be applied, with minor modifications, to any type of sample in which it is necessary to quickly detect the presence of the CagA antigen, or of more proteins encoded by the cag genes, such as blood, stool, gastric biopsies, gastric mucus or other fluids (e.g., water, in order to determine the environmental contamination, etc.).
- any type of sample in which it is necessary to quickly detect the presence of the CagA antigen, or of more proteins encoded by the cag genes such as blood, stool, gastric biopsies, gastric mucus or other fluids (e.g., water, in order to determine the environmental contamination, etc.).
- the present invention specifically provides an in vitro method for the rapid detection of infection from highly pathogenic Helicobacter pylori comprising the following steps:
- agglutination of said mixture shows the presence, in the biological sample under examination, of highly pathogenic Helicobacter pylori strains, which express the CagA protein or more proteins encoded by the cag genes.
- said biologi- cal sample may consist of gastric mucus, stools or of a biopsy specimen of the gastric mucosa.
- the above step a) consists of contacting a suspension of latex particles sensitized with an antiserum anti-total protein of CagA-positive H. pylori subsequently ad- sorbed with a strain of CagA-negative H. pylori with a biological sample to be analyzed for the presence of the CagA protein antigen or of protein antigens encoded by cag genes, and mixing said suspension with said biological sample.
- the visual detection b) is carried out with the aid of a positive control sample consisting of a suspension of CagA-positive H. pylori which is subjected, as the biological sample to be analyzed, to operations corresponding to steps a) and b).
- the visual detection b) is carried out with the aid of a negative control sample, consisting of a suspension of staphylococci or latex particles sensitized or not sensitized with hyperimmune serum, which is used in place of the suspension of the step a) when subjecting the biological sample to be analyzed to steps corresponding to the steps a) and b).
- the method according to the invention may further comprise the following steps:
- agglutination of said mixture shows the presence, in the biological sample under examination, of Helicobacter pylori strains
- the strain of Staphylococcus aureus Cowan 1 (ATCC 12598) is cultured overnight at 35- 37°C in tryptone-soy broth. Then, the culture is centrifuged and the sediment is washed two or three times with phosphate buffer (PBS, phosphate-buffered saline) at pH 7.2. After the last washing, the bacterial cells are suspended in PBS containing 0.5% formalin (37-40% formaldehyde in aqueous solution), and keept for about 3 hours at room temperature. Then, the cells treated with formalin are washed three or four times with PBS and the bacterial suspen- sion is adjusted to 10% in PBS. Finally, the suspension is heated to 80°C for one hour to obtain the inactivation of the bacteria.
- PBS phosphate buffer
- formalin 37-40% formaldehyde in aqueous solution
- 0.1 ml_ of undiluted antiserum is added to 1 ml_ suspension of staphylococci, and allowed to react for a sufficient time, for example, at least 30 minutes. Then, the suspension of sensitized staphylococci is diluted up to 10 ml_ with PBS and stored at 4°C until use.
- staphylococci may be replaced by latex particles of varying diameter to achieve a similar test of latex agglutination, the process of sensitization of the latex being similar to that for staphylococci.
- the anti-CagA serum to sensitize staphylococci, or latex particles may be obtained by directly using the CagA protein to immunize rabbits or other animals, or by adsorbing from the serum of a rabbit or other animal immunized with a strain of CagA-positive H. pylori the antibodies directed against helicobacter antigens, except the anti-CagA antibodies. This can be done by using a suspension of CagA-negative helicobacter: after adsorption, only anti-CagA antibodies remained in the serum.
- the tests according to the invention do not re- quire a reading carried out in laboratory: they are of immediate detection in the gastroenterologist's office. In practice, a quite evident agglutination is observed in less than one minute; in order to define a negative test, it necessary to wait for a couple of minutes.
- the negative control is represented by a suspension of staphylococci or latex particles not sensitized, or sensitized with non-specific immunoglobulins, which, obviously, must not agglutinate.
- the test proposed according to the invention can be carried out, in particular, both on samples of gastric mucus and in stool samples.
- the test is performed in the follow- ing way: a biopsy sample is dissolved in a drop of dithiothreitol, preferably 0.1 % in PBS (dithiothreitol has the ability to open the disulfide bridges of the mucoproteins, thus thinning the mucus); one drop of a 1 % suspension of staphylococci sensitized with anti-CagA antiserum is added and the mixture is stirrred with a stick.
- the test result is evaluated after waiting, preferably, for two minutes.
- the test is pref- erably performed with a negative control, which consists of a suspension of non-sensitized staphylococci, or of an iperimmune serum (with non-specific immunoglobulins) which, obviously, must not agglutinate when mixed with the test sample.
- a suspension of staphylococci or of latex particles sensitized with total antigens of H. pylori can also be added in the test, and suspensions of staphylococci or latex particles sensitized with an antiserum against total antigens of helicobac- ter can be colored with a predetermined color, while suspensions of staphylo- cocci or latex particles sensitized with anti-CagA antiserum can be colored with a different color.
- Staphylococci or latex particles sensitized with antiserum against total antigens of helicobacter may be colored, for example, in red, while those sensitized with anti-CagA antiserum may be colored in blue.
- the test according to the invention can be also carried out on samples of gastric mucus collected through a silk or cotton thread glued to a capsule that is swallowed; the capsule, as it proceeds along the esophagus and stomach, drags the thread behind it. The upper end of the thread is positioned on the cheek with a patch. Once arrived in the stomach, the capsule dissolves on contact with hydrochloric acid; after 10-15 minutes, the thread, strongly impregnated with mucus at the distal end, is retrieved; the mucus is recovered and the test is carried out
- the present invention concerns a diagnostic kit for the in vitro rapid diagnosis of infection by highly pathogenic strains of Helicobacter pylori, comprising the following components:
- A a suspension of staphylococci S. aureus Cowan 1 , or a suspension of latex particles, sensitized with anti-CagA antiserum, or with an antiserum against CagA-positive H. pylori from which the other antibodies against H. pylori antigens have been separated, in an aqueous solution such as phosphate-buffered saline (PBS) or PBS-formalin, containing an antimicrobial agent, such as merthiolate or sodium azide;
- PBS phosphate-buffered saline
- PBS-formalin an antimicrobial agent, such as merthiolate or sodium azide
- an aqueous solution such as PBS or PBS-formalin, containing a mucus-fluidifying agent, such as dithiothreitol, and an antimicrobial agent, such as m sodium erthiolate or sodium azide;
- a mucus-fluidifying agent such as dithiothreitol
- an antimicrobial agent such as m sodium erthiolate or sodium azide
- the proposed diagnostic kit also includes:
- D1 a suspension of CagA-positive H. pylori antigens, in an aqueous solution such as PBS or PBS-formalin, and an antimicrobial agent, such as merthioiate or sodium azide, as positive control for CagA-positive H. pylori.
- the proposed diagnostic kit also includes:
- D2. a suspension of H. pylori antigens, in an aqueous solution such as PBS or PBS-formalin, and an antimicrobial agent, such as merthioiate or sodium azide as a positive control for H. pylori.
- an antimicrobial agent such as merthioiate or sodium azide
- the component B contains dithiothreitol in a concentration comprised between 0.05 and 1 % by weight; in addition, said aqueous solution is a phosphate buffered saline (PBS) or a phosphate buffered saline containing 0.5-1.0% by weight of formalin (PBS-formalin), and said antimicrobial agent is merthioiate in a concentration of about 0.05% by weight or sodium azide in a concentration of about 0.1 % by weight Composition of the kit
- kits for the rapid diagnosis of H. pylori infection consist of the following elements:
- All mentioned components also contain a suitable concentration of merthiolate (thimerosal or, sodium ethylmercury-tiosalicilato), preferably to 0.05% by weight, or another equivalent antimicrobial agent.
- merthiolate thimerosal or, sodium ethylmercury-tiosalicilato
- the package contains the following:
- a flask of a third color for example red, containing total H. pylori antigens (for example, sonicated) in PBS and merthiolate, or in PBS-formalin (positive control for H. Pylori);
- Dithiothreitol can be used in concentrations from 0.05 to 1 % by weight, and is preferably 0.1 % by weight.
- dithiothreitol As an alternative to dithiothreitol, other known dissolvers of mucus may be used, such as those used as mucolytic agents in the pharmaceutical industry, including, in particular, the acetylcysteine.
- antimicrobial agents merthiolate can be used, for example, sodium azide, preferably 0.1 %.
- H. pylori As a positive control, in an alternative to suspension of H. pylori for- treated with formalin, it may be foreseen, according to the invention, the use of antigens of H. pylori extracts by means of ultrasonication or with glycine or with glucopiranoside (drastic detergent), in PBS containing sodium azide or merthiolate. Version on stool samples:
- the diagnostic kit according to the invention should also contain a vial containing PBS with an antimicrobial agent, preferably merthiolate, and a protease inhibitor (to prevent proteases from digesting protein antigens of H. pylori). After suspending a small amount of stool in this solution, the suspension is left to settle; then a small amount of supernatant is taken, which is used for the assay.
- an antimicrobial agent preferably merthiolate
- a protease inhibitor to prevent proteases from digesting protein antigens of H. pylori
- Figure 1 shows photographic images of the cardboards of a first ver- sion of the diagnostic kit according to the invention, with the results of the latex agglutination test for detection of strains of CagA-positive H. pylori, in which the following results are visible:
- wells 1 and 3 positive latex agglutination;
- the well 1 contains gastric mucus dissolved with dithiothreitol and mixed with a drop of latex sensitized with anti-CagA+ H. pylori;
- the well 3 contains a mixture of gastric mucus dissolved with dithiothreitol and mixed with a drop of latex sensitized with antibodies against total antigens of CagA+ H. pylori (positive control) - in the case shown the test is positive for CagA+H. pylori;
- well 2 contains a mixture of gastric mucus taken from a non-infected patient, in dithiothreitol, and sensitized latex; the test is negative;
- Figure 2 shows photographic images of the cardboards of a second preferred version of the diagnostic kit according to the invention, with the results of the latex agglutination test for the detection of high pathogenicity H. pylori strains with, in which the following results are visible: well 1 : agglutination obtained with latex sensitized with anti-CagA;
- wells 2 and 3 agglutination obtained with latex sensitized with antibodies anti-cag antigens
- well 4 negative control consisting of a cag-negative strain
- Figure 3 shows the photographic images of the cardboards of the second preferred embodiment of the diagnostic kit according to the invention, with the results of the latex agglutination test for the detection of high pathogenicity
- well 1 agglutination of a latex sensitized with antibodies anti-Cag anti- gens with two gastric biopsies from the same patient;
- well 2 a negative case, gastric mucus obtained from a patient a non- infected patient.
- the biopsy from the antrum and that from the body were diluted together in 100 pL of a 0.1 % solution of dithio- threitol in PBS pH 7.4 (to dissolve the gastric mucus); therefore, approximately 15 ⁇ of dissolved mucus was distributed on three different areas of a cardboard sheet with black background for agglutination. This operation was performed in duplicate, using two sets of six "wells" cardboard sheets.
- H. pylori was isolated in culture and/or observed under the microscope (true positives, TP); in the remaining 20 cases, culture and bacteriological examination were negative (true negatives, TN).
- LA latex agglutination test
- the 1-2% of patients hosts the coccoid form of H. pylori in the stomach, which retains its antigenicity (and therefore can be highlighted by the LA test), but cannot be cultured, and is not detectable by histologic examination.
- a serum obtained as in the case No. 2 contains a much greater concentration IgGs against virulence antigens, compared to serum No. 1 , which was obtained by immunizing rabbits with the recombinant CagA protein only.
- the wild CagA+ strains of possess an insertion called "pathogenicity island”; that contains about thirty genes involved in virulence, and cagA is only one of these.
- These genes encode peptides with many different functions, which, overall, are used by the bacterium to move the CagA protein in the colonized gastric epithelial cell.
- These peptides are all immunodominant determinants and stimulate, in the infected individual and in immunized rabbits, a strong production of antibodies.
- a serum anti-total proteins of a CagA+ strain, adsorbed with a suspension of a CagA " strain will contain many more antibodies, all directed against antigens involved in virulence.
- the animal was immunized intravenously (through the marginal ear vein) with seven doses of a suspension of strain G33, each consisting of approximately 10 10 microorganisms per ml in PBS with 0.5% formalin.
- the im- munization was carried out every 3-4 days. After the V dose, small bleedings were performed and the rabbit was finally deprived of all the blood that was possible to obtain (about 50 ml), and then the rabbit was sacrificed when the level of serum agglutinating antibodies had reached the value 1 :2000. After sensitizing latex with the serum obtained, and after testing the sensitivity and specificity thereof, the anti-G33 serum was adsorbed with the H. pylori G21 strain, non cytotoxic and free of the cag island (Xiang et al., ibidem).
- the adsorption was carried out in the following way: G21 was inoculated on ten agar-blood plates, which were incubated in a microaerobic atmosphere at 37°C for 72 hours. The bacterial film was then collected and washed twice with PBS; 1 g of wet paste was then mixed thoroughly with 0.5 ml of anti-G33 serum (final dilution 1 :4 in PBS). The mixture was placed to incubate in normal air at 37°C for two hours, with frequent agitation in Vortex; subsequently, it was placed to incubate at 4°C overnight (in order to allow adsorption of incomplete antibodies).
- the mixture was centrifuged at 4000 g at 4°C for 20 min and the adsorbed serum was collected and used for sensitizing a latex suspension.
- the latex so sensitized was then used to identify cag positive strains cultures on plate and in gastric biopsies.
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Abstract
The invention relates to an in vitro method for the detection of the pres- ence of highly pathogenic CagA+ strains of Helicobacter
pylori, or of H. pylori strains expressing the cag pathogenicity island, in samples of gastric mucus, feces or biopsies, which method provides results immediately available on performing the test, as well as the diagnostic kits for use in carrying out said method. The method exploits a coagglutination or latex agglutination test pro- cedure implemented by sensitizing with anti-CagA sera or with an antiserum anti-total protein of CagA-positive H. pylori from which the other antibodies against antigens of H. pylori have been separated, with a suspension of staphylococci with a high content of Protein A or with latex particles, and de- tecting the possible agglutination in biological samples examined.
Description
METHOD FOR RAPID DETECTION OF INFECTION FROM CagA POSITIVE HELICOBACTER PYLORI AND DIAGNOSTIC KIT THEREFOR
DESCRIPTION
Field of the invention
The present invention concerns a method for the rapid detection of infection from CagA-positive Helicobacter pylori, and a diagnostic kit for carrying out said method. More specifically, the invention concerns an in vitro method for detecting, in samples of gastric mucus, stools or biopsy specimens, the presence of highly pathogenic CagA strains of H. pylori, which method provides results immediately available on performing the test, as well as the diagnostic kits for use in carrying out said method. Background of the invention
As is known, Helicobacter pylori is a helix-shaped Gram-negative mi- croaerophilic microorganism that can inhabit the stomach and the duodenum, adapting itself to the inhospitable environment of the gastric mucosa in a quite particular way, to the point that it often fails to produce any detectable symp- toms. Actually, it is considered that more than 80% of the individuals infected with H. pylori are asymptomatic. Such microorganism infects more than half of the world population at the level of the gastro-duodenal tract. The transmission route of the infection has not been clarified, although infection seems to be typically acquired in the childhood.
The stomach is protected from its own gastric juice, made of concentrated hydrochloric acid and rich of digestive enzymes, by a thick layer of mucus which covers the gastric mucosa, within which the Helicobacter may penetrate and survive, resisting to any acid that may possibly reach it thanks to an enzyme produced by such bacterium, urease. Urease converts the urea contained in large amounts in the stomach into bicarbonate and ammonium, therefore a basic environment is established in the vicinity of the bacterial colony, such environment being capable of neutralizing the hydrochloric acid of
gastric secretions, thereby protecting H. pylori.
Another protective mechanism available to Helicobacter pylori consists in that the natural immune defences of the body cannot reach the microorganism in the gastric mucus. The immune system reacts to H. pylori infec- tion by sending leukocytes, T killer lymphocytes and other defence factors, but these cannot reach the infection site, as they cannot easily penetrate through the mucus layer, and remain and accumulate in the site, leaving there their destructive content (consisting of superoxide radicals) when they die. Additional nutrients are sent on the site to assist the white blood cells, and this fact does no more than further feeding Helicobacter itself. As a result, gastritis develops in a few days, and then it may sometimes progress up to peptic ulcer. In view of the foregoing, it is considered that the causative agent of the damage to the gastric mucosa is not H. pylori itself, but the inflammation and, consequently, the immune response to the microorganism.
From the scientific literature it appears that infection from Helicobacter pylori is the main cause of chronic gastritis and the most relevant factor for the development of peptic ulcer (Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983; 1 :1273-5). By colonizing the stomach, H. pylori induces a persistent inflammation (chron- ic gastritis) which may last years without developing with clinical symptoms. However, in 10-20% of the cases, chronic gastritis may result in the development of gastric or duodenal ulcers.
Also gastric cancer (gastric adenocarcinoma and non-Hodgkin gastric lymphoma) is often associated with H. pylori (Moss SF. The carcinogenic ef- feet of H. pylori on the gastric epithelial cell. J Physiol Pharmacol 1999; 50:847-56). In the frame of a wide review of cases of gastric carcinoma it has been shown that the presence of H. pylori results in a six-fold increase of the risk of gastric cancer. It is believed that chronic gastritis may lead to intestinal metaplasia, which in turn, may degenerate into malignant cancer. The low grade malignant lymphoma o MALToma, in turn, seems to derive from the malignant degeneration of lymphoid tissue associated with the mucosa. In this case, retrospective bioptic studies demonstrated that 90% of these
MALTomes are associated to the presence of H. pylori.
In addition to the mentioned gastroduodenal diseases, infection with H. pylori can aggravate or contribute to the development of coronary heart disease (ischemic heart disease up to myocardial infarction), metabolic disorders (such as increased LDL cholesterol and HDL cholesterol reduction, insulin resistance, etc.), autoimmune diseases (such as autoimmune thyroiditis, systemic sclerosis, etc.) and several other conditions such as osteoporosis, reduced male and female fertility, etc.. For all these reasons, it is necessary to detect early infection and treat it appropriately.
The diagnosis of H. pylori infection is commonly done through the research of bacterial urease in gastric biopsies, during endoscopy, or by looking for bacterial antigens in stool samples. Gastrointestinal endoscopic examination is certainly more reliable because it allows direct viewing of esophageal and gastric mucosa, removal of material by biopsy and culture of the material removed for H. pylori. However, it is an invasive and rather expensive test.
The search of anti-Hp antibodies in blood would represent a cheap and fairly rapid method of diagnosis, advantageous due to the fact that the detection of specific IgGs in serum has good sensitivity and good specificity. However, its utility is severely limited, as it does not distinguish between active in- fection and past infection. In fact, the anti-helicobacter antibodies can circulate in the bloodstream for more than two years after recovery from the infection.
The search for fecal antigen of H. pylori, HpSA, may identify the presence of antigen in stool and, since this is a sign of an active infection, this test is more reliable than detection of antibodies in the blood. However, with this type of survey the level of false negatives is remarkable, therefore the test is safe only when it is positive. A known method of rapid diagnosis based on detection of the fecal H. pylori antigen employs an analytical technique known as coagglutination test, in its variant known as "latex agglutination", which uses polymeric latex particles.
The original method of coagglutination (Kronvall, G. 1973. A rapid slide agglutination method for typing pneumococci by means of specific antibody absorbed to protein A-containing staphylococci. J. Med. Microbiol. 6: 187-190)
is based on a Staphylococcus strain, the strain of Staphylococcus aureus Cowan 1 , which is rich in protein A, a substance having the characteristic of adsorbing in a specific, but non-immunological, manner, immunoglobulins G (IgGs) of subclasses 2 and 4, linking the Fc fragment and leaving the Fab por- tion free to interact with a variety of antigenic molecules. Staphylococci coated with antibodies thus become a reagent which agglutinates in the presence of the homologous antigen. Agglutination of this S. at/ret/s-protein A complex can be used to detect the presence of antigens (bacterial, viral and parasitic antigens), in body fluids, and for the immunological identification of various pathogens.
In the variant known as latex agglutination test staphylococci are replaced by polymer microspheres of uniform size (typically with a diameter of 8 μιτι) obtained by emulsion polymerization of hydrocarbon monomers. The microspheres, known in the field with the name of "latex" particles, exhibit, on their surface, the hydrophilic heads of the hydrocarbon chains that constitute them. These are coated with antisera of bacterial, viral or other origin in a manner similar to that described for staphylococci suspensions, and then the latex sensitization process is similar to that previously described for staphylococci containing Protein A. Therefore, the rapid diagnostic method mentioned above (Blanco et al., "Evaluation of a latex agglutination test (PYLOGEN) for the detection of Helicobacter pylori in stool specimens", Diagn Microbiol Infect Dis. 2009; 63: 349-353) is based on putting in contact a suspension of latex particles sensitized with a monoclonal antibody directed specifically against H. pylori antigens with a stool sample of the subject to be analyzed, and visually checking the possible agglutination, which would be indicative of the presence of H. pylori in the sample.
It should be noted that the H. pylori antigens used for sensitization of the latex particles in the test mentioned above are of polysaccharide nature. According to current knowledge, it is presumed that the different H. pylori strains will not be able to differentiate between fully virulent (because associated with ulcers and cancer) and less virulent on the basis of the constitution of their polysaccharide. In addition, as is evident from the work mentioned
above, the main indication of Pylogen is the verification of the eradication treatment, because its sensitivity (75%) in the post-treated patient is slightly better than that obtained in the pre-treatment diagnosis (73%), although the specificity compared to UBT (Urea Breath Test, described below) is less good (93% vs. 100%).
The examination for H. pylori infection currently regarded as the "gold standard" is the breath test with 13C-labeled urea (Urea Breath Test), which is still performed in a few centers, mainly because of its cost. The examination must be performed after an overnight fast, and involves the oral assumption of sodium citrate, followed, after ten minutes, by an examination of the air exhaled by the patient. The patient is then administered one tablet of radio- labelled urea, and a new sample of exhaled air is collected after about 30 minutes. Since, as noted, H. pylori splits urea into ammonia and bicarbonate, the amount of marked CO2 exhaled after half an hour from a subject with on- going infection will be higher than that exhaled from a healthy subject.
It is also known that the various strains of Helicobacter pylori do not all have the same pathogenic power: the strains having in their chromosome an insertion defined "cag pathogenicity island" (cag PAI), a set of genes involved in the virulence of the microorganism, exhibit a higher inflammatory potential. Approximately 50-70% of the strains of H. pylori found in Western countries contain the cag pathogenicity island, and patients infected with bacteria whose genome contains the insertion have a stronger gastric inflammatory response and a greater risk of developing peptic ulcers or gastric cancer than patients infected with strains that do not contain this insertion into their genome. The cag-positive strains are able to inject an oncoprotein (encoded by one of the cag genes) into colonized gastric cells, named CagA, which increases the risk of developing neoplastic lesions (Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, et al. cag, a pathogenicity island of Helicobacter pylori, encodes type l-specific and disease-associated virulence factors. Proc Natl Acad Sci USA 1996; 93:14648-53).
Actually, it is to be considered that H, pylori is a microorganism that has evolved with man. It is part of the genus Helicobacter with thirty different spe-
cies that have found their habitat in the stomach of many mammals and in intestines of many vertebrates, mammals and not (e.g. birds). The presence of H. pylori had to confer a selective advantage to individuals hosting it, otherwise this organism would have been lost in the evolutionary path. It is believed that hundreds of thousands of years ago, H. pylori was infected by a phage which inserted in its chromosome a DNA fragment taken from a bacterial species that no longer exists (because the nucleotide sequence of this segment is not found in any of the microorganisms examined so far). The insertion contains 30 genes involved in virulence, and is called "pathogenicity island" (PAI).
Other PAIs were already known in various bacterial species infecting humans and plants, such as Yersinia enterocolitica subsp. pestis, Bordetella pertussis, Agrobacterium tumefaciens: the genes which make up the various PAIs show a very high homology with each other, and all encode for the production of a species of conjugation factor, through which CagA, a protein con- sidered to be an immune-dominant determinant, it is translated, almost injected, into the eukaryotic cell colonized. Once inside the cell, the CagA protein is phosphorylated in its tyrosine residues and transformed into an oncoprotein, able to alter the cell morphology and thus disturb the "signaling" systems of the host in such a way that can lead, ultimately, to the development of a gas- trie carcinoma. The carcinogenicity of CagA is demonstrated by the observation that transgenic mice expressing CagA systemically develop gastric carcinoma or tumors of the hematopoietic system.
In addition to the CagA protein, the bacterium also transfers part of its peptidoglycan, a constituent of its wall which has the ability to stimulate a strong inflammatory response mediated by transduction of the signal to which NF-πΒ participates. Inflammatory damage is added to the damage caused by CagA (which also has a strong pro-inflammatory power), and this combined action explains why patients with stomach carcinoma are, or were, all, with very few exceptions, infected with strains of H. pylori expressing CagA.
Studies on the polymorphism of the CagA protein have also shown that strains with mutilating deletions of the PAI or with few genomic repetitions of the variable region of cagA (encoding the amino acid sequence Epiya - Glu-
Pro-lle-Tyr-Ala - which is the one that is phosphorylated) are isolated less frequently in cases of gastric carcinoma, but rather increase the risk of peptic ulcer disease. The discovery of CagA-negative strains of H. pylori is exceptional in patients with ulcer or cancer. In other words, strains which host the PAI, of which CagA is the marker, can determine both peptic ulcer and gastric carcinoma. What makes the difference, apart from the host's inflammatory haplo- type, is the polymorphism of cagA and/or the PAI genotype.
The foregoing observations have almost immediate practical effects, as they suggest that the diagnosis of H. pylori infection, as currently performed, is insufficient and incomplete in the absence of a characterization of the infecting strain CagA status. In fact, the risk of such diseases is high if the infecting bacteria host CagA, and is low if they are cagA-free.
As to the effects of CagA on extra-gastric diseases, the following observations have recently been made (Figura N, Franceschi F, Santucci A, Bernardini G, Gasbarrini G, Gasbarrini A. Extragastric manifestations of Helicobacter pylori infection. Helicobacter, 2010 Sep.15 Suppl 1 :60-8):
1 . Ischemic heart disease. Infection with CagA-positive strains is significantly more frequent in patients than in controls without cardiovascular disorders. The patients infected with CagA-positive strains also have higher cardiovascular damage, as evidenced by stress ECG, and serum levels of BNP (B-type Natriuretic Peptide) significantly higher (Figura N, Palazzuoli A, Vaira D, Campagna M, Moretti E, lacoponi F, Giordano N, Clemente S, Nuti R, Ponzetto A. Cross-sectional study: CagA-positive Helicobacter pylori infection, acute coronary artery dis- ease and systemic levels of B-type natriuretic peptide. J Clin Pathol.
2014 Mar; 67(3):251-7).
2. Autoimmune thyroid disease. Infection with CagA-positive H. pylori is more prevalent in patients than in controls; moreover, it was also shown that eradication of the infection determines the disappearance of anti-thyroglobulin and anti-peroxidase antibodies.
3. The transaminases are higher (but still within the normal range) in patients who host a strain expressing CagA.
4. Patients with duodenal ulcers have higher concentrations of monocytes and polymorphonucleates if they have anti-CagA antibodies.
5. The frequency of infection by CagA-positive strains is higher in patients with osteoporosis than in controls, and anti-CagA antibody titers corre- late in an almost linear fashion with the risk of pathological fractures in these patients.
6. Patients and individuals with anti-CagA antibodies are significantly more frequently infertile, both male and females, and males have a reduced percentage of sperm with reduced motility, and a high level of systemic inflammatory cytokines (Moretti E, Collodel G, Mazzi L, Cam- pagna MS, Figura N. CagA-positive Helicobacter pylori infection and reduced sperm motility, vitality, and normal morphology. Dis Markers. 2013; 35(4):229-34).
Considering the diagnostic methods currently in use to detect the infec- tion by Helicobacter pylori, the serologic method is, at the present time, the only method able to evidence an infection by the most virulent strains, since it allows to detect the presence of anti-CagA antibodies in the blood. However, as these antibodies can continue to circulate in the blood for as long as 10 years before going back to levels below the detection threshold of the com- mon commercial tests, it is evident that a diagnosis of H. pylori infection allowing to detect high risk strains when the infection is in place is not possible through this method.
In the light of the foregoing, it emerges that there does not exist, to date, a method suitable to detect the presence of H. pylori strains expressing CagA, or to detect the only CagA protein antigen, in biological samples taken from patients in an extemporaneous test, or in a test being quickly performa- ble and having good reliability. The availability of such a diagnostic method would allow to carry out a rapid screening in order to identify which subjects, among those infected with H. pylori in place, are affected by CagA-positive strains, and thus are at higher risk of critical development of the pathology.
Summary of the invention
According to the present invention, it has been found that it is possible to rapidly discriminate, and with great sensitivity, between infections from CagA negative H. pylori and infections from CagA-positive H. pylori by apply- ing the known analytical technique known as coagglutination test, both in its variant employing the Staphylococcus aureus Cowan 1 strain, and in its variant employing polymeric latex particles.
In the frame of the studies that led to the present invention, the possibility of sensitizing a latex polymer with an anti-CagA serum obtained by immun- izing rabbits with a recombinant fragment from the CagA protein, whose gene had been cloned in Escherichia coli, was first considered. This method, although specific, did not prove to be very sensitive, probably due to the low concentration of antibodies in the serum.
In fact, the number and concentration of antigens required to produce a hyperimmune serum are both important factors, as it is also demonstrated by the observation that the latex sensitization with purified IgGs obtained from rabbits immunized with total protein antigens of H. pylori gives rise to a quick and highly visible agglutination when the amount of IgGs used is adequate, but if the amount of IgGs is just halved the quantity of IgGs sensitizing aggluti- nation is considerably less evident.
In order to overcome the problem of a not optimal sensitivity of the above-described method which uses latex particles sensitized with anti-CagA antiserum, purified IgGs anti-total protein of H. pylori were produced, which were subsequently adsorbed with a suspension of a non-cytotoxic CagA- negative H. pylori strain. In this way, the serum was deprived of antibodies directed against antigens different from CagA and from other peptides encoded by the genes of the pathogenicity island (about 30) located upstream from cagA. It has been considered, in fact, that CagA is only one of the proteins encoded by the genes of the pathogenicity island cag (which contains about 30 "open reading frames"), each of which encodes for a peptide provided, as CagA, with a high immunogenicity. Furthermore, studies performed in the 90's have shown that, in a relevant portion of the isolated strains, cag is composed
by two sections, right and left with respect to an insertion known as IS605, which divides it into two parts without interrupting it. Its presence causes instability of cag, resulting into a possible deletion of an arm of the island. These deletions reduce the virulence of the strains; it happens for some point muta- tions of genes located in the left or right branch, such as cagE and virBIO.
As an evidence of the foregoing, a study conducted by the research group of the author of the present invention showed that the long-term prognosis (15 years) of cases of gastric cancer is much better when the infecting strain is free from the cagE and/or virBIO genes.
Using in the coagglutination test an antiserum against total proteins of
CagA+ H. pylori deprived of the other antibodies against H. pylori antigens different from those which characterize the pathogenicity island, in particular obtained by adsorbing the anti-total protein of CagA+ H. pylori antiserum with a suspension of a CagA-negative strain of H. pylori, it was possible to obtain, according to the invention, a good agglutination, equally specific not only for the CagA protein, but also for all other high virulence proteins of the cag pathogenicity island of H. pylori Type I (CagA positive and cytotoxic).
According to the present invention there is provided, therefore, a rapid diagnostic method for the detection of H. pylori strains expressing the cag pathogenicity island genes, particularly CagA-positive H. pylori strains, which method exploits the method of coagglutination with S. aureus or the method of latex agglutination, in which staphylococci or latex particles sensitized with an- ti-CagA antiserum, or with an antiserum anti-total proteins of CagA-positive H. pylori from which other antibodies against H. pylori antigens are contacted, in suitable conditions, with a biological sample to be analyzed, and the agglutination or not of the resulting suspension is detected, according to whether or not CagA antigens, and other high virulence proteins of the cag pathogenic island, are present in the sample
The proposed method according to the invention can be applied, with minor modifications, to any type of sample in which it is necessary to quickly detect the presence of the CagA antigen, or of more proteins encoded by the cag genes, such as blood, stool, gastric biopsies, gastric mucus or other fluids
(e.g., water, in order to determine the environmental contamination, etc.).
Detailed description of the invention
Therefore, the present invention specifically provides an in vitro method for the rapid detection of infection from highly pathogenic Helicobacter pylori comprising the following steps:
a) contacting a suspension of staphylococci S. aureus Cowan 1 , or a suspension of latex particles, sensitized with anti-CagA antiserum, or with an antiserum anti-total protein of CagA-positive H. pylori from which the other an- tibodies against H. pylori antigens have been separated, with a biological sample to be analyzed for the presence of the CagA protein antigen, or of protein antigens encoded by the cag genes of H. pylori, and mixing said suspension with said biological sample;
b) visually detecting the possible agglutination of the mixture resulting from the previous step;
wherein the agglutination of said mixture shows the presence, in the biological sample under examination, of highly pathogenic Helicobacter pylori strains, which express the CagA protein or more proteins encoded by the cag genes.
According to some specific embodiments of the invention, said biologi- cal sample may consist of gastric mucus, stools or of a biopsy specimen of the gastric mucosa.
According to some preferred embodiments of the invention, the above step a) consists of contacting a suspension of latex particles sensitized with an antiserum anti-total protein of CagA-positive H. pylori subsequently ad- sorbed with a strain of CagA-negative H. pylori with a biological sample to be analyzed for the presence of the CagA protein antigen or of protein antigens encoded by cag genes, and mixing said suspension with said biological sample.
Preferably, according to the invention, the visual detection b) is carried out with the aid of a positive control sample consisting of a suspension of CagA-positive H. pylori which is subjected, as the biological sample to be analyzed, to operations corresponding to steps a) and b).
According to other preferred embodiments of the invention, the visual detection b) is carried out with the aid of a negative control sample, consisting of a suspension of staphylococci or latex particles sensitized or not sensitized with hyperimmune serum, which is used in place of the suspension of the step a) when subjecting the biological sample to be analyzed to steps corresponding to the steps a) and b).
In the specific case in which it is also requested to simultaneously detect the presence in the sample of any generic H. pylori strains, the method according to the invention may further comprise the following steps:
c) contacting a suspension of staphylococci S. aureus Cowan 1 , or a suspension of latex particles, sensitized with antiserum against total H. pylori antigens, with a biological sample to be analyzed for the presence of H. pylori antigens, and mixing said suspension with said biological sample; and
d) visually detecting the possible agglutination of the mixture resulting from the previous operation;
wherein the agglutination of said mixture shows the presence, in the biological sample under examination, of Helicobacter pylori strains, and
wherein the combined performance of the mentioned steps a), b, c) and d) allows to detect both a possible infection by H. pylori and the possible presence of highly pathogenic Helicobacter pylori strains.
According to a specific embodiment of the diagnostic method of the invention, in the version that uses coagglutination with S. aureus, the strain of Staphylococcus aureus Cowan 1 (ATCC 12598) is cultured overnight at 35- 37°C in tryptone-soy broth. Then, the culture is centrifuged and the sediment is washed two or three times with phosphate buffer (PBS, phosphate-buffered saline) at pH 7.2. After the last washing, the bacterial cells are suspended in PBS containing 0.5% formalin (37-40% formaldehyde in aqueous solution), and keept for about 3 hours at room temperature. Then, the cells treated with formalin are washed three or four times with PBS and the bacterial suspen- sion is adjusted to 10% in PBS. Finally, the suspension is heated to 80°C for one hour to obtain the inactivation of the bacteria.
To sensitize staphylococci, 0.1 ml_ of undiluted antiserum is added to 1
ml_ suspension of staphylococci, and allowed to react for a sufficient time, for example, at least 30 minutes. Then, the suspension of sensitized staphylococci is diluted up to 10 ml_ with PBS and stored at 4°C until use.
As already noted, staphylococci may be replaced by latex particles of varying diameter to achieve a similar test of latex agglutination, the process of sensitization of the latex being similar to that for staphylococci.
The anti-CagA serum to sensitize staphylococci, or latex particles, may be obtained by directly using the CagA protein to immunize rabbits or other animals, or by adsorbing from the serum of a rabbit or other animal immunized with a strain of CagA-positive H. pylori the antibodies directed against helicobacter antigens, except the anti-CagA antibodies. This can be done by using a suspension of CagA-negative helicobacter: after adsorption, only anti-CagA antibodies remained in the serum.
It should be noted that the tests according to the invention do not re- quire a reading carried out in laboratory: they are of immediate detection in the gastroenterologist's office. In practice, a quite evident agglutination is observed in less than one minute; in order to define a negative test, it necessary to wait for a couple of minutes. The negative control is represented by a suspension of staphylococci or latex particles not sensitized, or sensitized with non-specific immunoglobulins, which, obviously, must not agglutinate.
The test proposed according to the invention can be carried out, in particular, both on samples of gastric mucus and in stool samples.
Version on gastric mucus:
According to a specific embodiment, the test is performed in the follow- ing way: a biopsy sample is dissolved in a drop of dithiothreitol, preferably 0.1 % in PBS (dithiothreitol has the ability to open the disulfide bridges of the mucoproteins, thus thinning the mucus); one drop of a 1 % suspension of staphylococci sensitized with anti-CagA antiserum is added and the mixture is stirrred with a stick.
If the sample contains CagA-positive helicobacter, a very apparent agglutination is observed in less than one minute. The test result is evaluated after waiting, preferably, for two minutes. For greater certainty, the test is pref-
erably performed with a negative control, which consists of a suspension of non-sensitized staphylococci, or of an iperimmune serum (with non-specific immunoglobulins) which, obviously, must not agglutinate when mixed with the test sample.
According to some preferred embodiments of the proposed method, a suspension of staphylococci or of latex particles sensitized with total antigens of H. pylori can also be added in the test, and suspensions of staphylococci or latex particles sensitized with an antiserum against total antigens of helicobac- ter can be colored with a predetermined color, while suspensions of staphylo- cocci or latex particles sensitized with anti-CagA antiserum can be colored with a different color. In this way, both the infection and the genotype of Helicobacter are diagnosed with a single test. Staphylococci or latex particles sensitized with antiserum against total antigens of helicobacter may be colored, for example, in red, while those sensitized with anti-CagA antiserum may be colored in blue.
The test according to the invention can be also carried out on samples of gastric mucus collected through a silk or cotton thread glued to a capsule that is swallowed; the capsule, as it proceeds along the esophagus and stomach, drags the thread behind it. The upper end of the thread is positioned on the cheek with a patch. Once arrived in the stomach, the capsule dissolves on contact with hydrochloric acid; after 10-15 minutes, the thread, strongly impregnated with mucus at the distal end, is retrieved; the mucus is recovered and the test is carried out
According to a further aspect thereof, the present invention concerns a diagnostic kit for the in vitro rapid diagnosis of infection by highly pathogenic strains of Helicobacter pylori, comprising the following components:
A. a suspension of staphylococci S. aureus Cowan 1 , or a suspension of latex particles, sensitized with anti-CagA antiserum, or with an antiserum against CagA-positive H. pylori from which the other antibodies against H. pylori antigens have been separated, in an aqueous solution such as phosphate-buffered saline (PBS) or PBS-formalin, containing an antimicrobial agent, such as merthiolate or sodium azide;
B. an aqueous solution such as PBS or PBS-formalin, containing a mucus-fluidifying agent, such as dithiothreitol, and an antimicrobial agent, such as m sodium erthiolate or sodium azide;
C. some cardboard sheets with dark disks, on which the test is to be per- formed.
According to a preferred embodiment of the invention, the proposed diagnostic kit also includes:
D1. a suspension of CagA-positive H. pylori antigens, in an aqueous solution such as PBS or PBS-formalin, and an antimicrobial agent, such as merthioiate or sodium azide, as positive control for CagA-positive H. pylori.
According to a further preferred embodiment of the invention, the proposed diagnostic kit also includes:
D2. a suspension of H. pylori antigens, in an aqueous solution such as PBS or PBS-formalin, and an antimicrobial agent, such as merthioiate or sodium azide as a positive control for H. pylori.
Preferably, in the proposed diagnostic kit according to the invention, the component B contains dithiothreitol in a concentration comprised between 0.05 and 1 % by weight; in addition, said aqueous solution is a phosphate buffered saline (PBS) or a phosphate buffered saline containing 0.5-1.0% by weight of formalin (PBS-formalin), and said antimicrobial agent is merthioiate in a concentration of about 0.05% by weight or sodium azide in a concentration of about 0.1 % by weight Composition of the kit
A specific embodiment of a kit for the rapid diagnosis of H. pylori infection consists of the following elements:
a suspension in PBS of staphylococci or latex sensitized with antiserum against CagA-positive H. pylon or with an antiserum against CagA- positive H. pylori from which the other antibodies against antigens of H. pylori have been separated;
a PBS solution at pH 7.4 with dithiothreitol (or other mucus-thinning
agent) (to avoid contamination and kill H. pylori);
a suspension of CagA-positive H. pylori treated with formalin as positive control for highly pathogenic strains (with anti-CagA antibodies).
All mentioned components also contain a suitable concentration of merthiolate (thimerosal or, sodium ethylmercury-tiosalicilato), preferably to 0.05% by weight, or another equivalent antimicrobial agent.
According to a preferred embodiment of the diagnostic kit proposed according to the invention, the package contains the following:
• a flask of a first color, for example blue, with a suspension of staphylococci with anti-CagA abtibodies in PBS, with merthiolate as preservative (test suspension);
• a flask of a second color, for example white, with a solution of 0.1 % di- thiothreitol in PBS with merthiolate as preservative (mucus thinner suspension);
• a flask of a third color, for example red, containing total H. pylori antigens (for example, sonicated) in PBS and merthiolate, or in PBS-formalin (positive control for H. Pylori);
' some cardboard sheets with dark disks, on which the test is to be performed..
Dithiothreitol can be used in concentrations from 0.05 to 1 % by weight, and is preferably 0.1 % by weight.
As an alternative to dithiothreitol, other known dissolvers of mucus may be used, such as those used as mucolytic agents in the pharmaceutical industry, including, in particular, the acetylcysteine.
As antimicrobial agents merthiolate can be used, for example, sodium azide, preferably 0.1 %.
As a positive control, in an alternative to suspension of H. pylori for- treated with formalin, it may be foreseen, according to the invention, the use of antigens of H. pylori extracts by means of ultrasonication or with glycine or with glucopiranoside (drastic detergent), in PBS containing sodium azide or merthiolate.
Version on stool samples:
In the case of tests to be performed on stool samples, the diagnostic kit according to the invention should also contain a vial containing PBS with an antimicrobial agent, preferably merthiolate, and a protease inhibitor (to prevent proteases from digesting protein antigens of H. pylori). After suspending a small amount of stool in this solution, the suspension is left to settle; then a small amount of supernatant is taken, which is used for the assay.
The specific features of the invention, as well as the advantageous aspects thereof, will become more apparent with reference to the detailed de- scription presented merely by way of example in the following, together with the results of the experimentation carried out on it.
Brief description of the drawings
Figure 1 shows photographic images of the cardboards of a first ver- sion of the diagnostic kit according to the invention, with the results of the latex agglutination test for detection of strains of CagA-positive H. pylori, in which the following results are visible:
wells 1 and 3: positive latex agglutination; the well 1 contains gastric mucus dissolved with dithiothreitol and mixed with a drop of latex sensitized with anti-CagA+ H. pylori; the well 3 contains a mixture of gastric mucus dissolved with dithiothreitol and mixed with a drop of latex sensitized with antibodies against total antigens of CagA+ H. pylori (positive control) - in the case shown the test is positive for CagA+H. pylori;
well 2 contains a mixture of gastric mucus taken from a non-infected patient, in dithiothreitol, and sensitized latex; the test is negative;
wells from 4 to 6 contain sensitized latex and an amount of agar culture of Helicobacter felis, Helicobacter mustelae and Campylobacter jejuni respectively: tests are negative, demonstrating the specificity of the reaction.
Figure 2 shows photographic images of the cardboards of a second preferred version of the diagnostic kit according to the invention, with the results of the latex agglutination test for the detection of high pathogenicity H. pylori strains with, in which the following results are visible:
well 1 : agglutination obtained with latex sensitized with anti-CagA;
wells 2 and 3: agglutination obtained with latex sensitized with antibodies anti-cag antigens;
well 4: negative control consisting of a cag-negative strain;
Figure 3 shows the photographic images of the cardboards of the second preferred embodiment of the diagnostic kit according to the invention, with the results of the latex agglutination test for the detection of high pathogenicity
H. pylori strains, in which the following results are visible:
well 1 : agglutination of a latex sensitized with antibodies anti-Cag anti- gens with two gastric biopsies from the same patient;
well 2: a negative case, gastric mucus obtained from a patient a non- infected patient.
Latex test of gastric biopsies for the diagnosis of infection with Helico- bacter pylori expressing CagA
I . Latex sensitized with anti-CagA antiserum
Patients and Methods
41 patients who underwent esophagogastroduodenoscopy for dyspep- sia were tested. From each patient four biopsies from the antrum - for the latex agglutination, the rapid urease test, the standard culture and histology, and the modified Giemsa stain (for bacteriological examination) and three biopsies from the body/fundus - for latex agglutination, culture and histolo- gy/bacterioscopic examination were taken. A sample of blood from which the serum was separated was also taken from patients, the sample being stored at -20°C until the time of the assay. The sera of patients found to be infected have been then examined for the presence of anti-CagA antibodies with a commercially available ELISA method: "CagA IgG ELISA kit", provided by Genesis Diagnostics, Littleport, UK (96% sensitivity and 97% specificity, inter- assay coefficient of variation <12%).
For the execution of the latex test, the biopsy from the antrum and that from the body were diluted together in 100 pL of a 0.1 % solution of dithio-
threitol in PBS pH 7.4 (to dissolve the gastric mucus); therefore, approximately 15 μΙ of dissolved mucus was distributed on three different areas of a cardboard sheet with black background for agglutination. This operation was performed in duplicate, using two sets of six "wells" cardboard sheets.
To the first sample of the first set of cardboard sheets a drop of sensitized latex containing antibodies to surface antigens of H. pylori (LHP) was added; to the second sample one drop of LHP and a drop of a suspension of H. pylori CCUG 17874 (type strain, CagA-positive) containing 106 organisms per ml in PBS with 1 % formalin (positive control) was added, and to the third sample a drop of latex sensitized with rabbit hyperimmune serum (negative control) was added, and mixed thoroughly with a stick. In case of positivity, a clear agglutination was observed in the first two reaction areas, in less than a minute. The negative control remained such even after five minutes.
Then the determination of the presence of the CagA antigen in the sec- ond series of cardboard sheets was carried out. The procedure was the same, with the difference that instead of LHP, a suspension of sensitized latex containing anti-CagA antibodies (LCA) was used.
Results
In 21 out the 41 cases examined, H. pylori was isolated in culture and/or observed under the microscope (true positives, TP); in the remaining 20 cases, culture and bacteriological examination were negative (true negatives, TN).
The latex test according to the invention resulted to be positive in 20 of the 21 TP cases (sensitivity = 95.2%) and negative in 18 of the 20 TN cases (specificity = 90.0%), as shown in Table 1 below.
!e 1. Results of tests with sensitized latex containing antibodies against to- antigens of H. pylori (LHP)
Of the 20 cases in which the latex test was positive for the presence of H. pylori, the serological test for anti-CagA antibodies was positive in 12 cases (CTP), and negative in 8 cases (CTN). The latex test for anti-CagA was positive in 10 cases (sensitivity = 83.3%), negative in 7 cases (specificity = 87.5%) as shown in Table 2 below. Table 2. Results of tests with sensitized latex containing anti-CagA antibodies (LCA)
Unpublished data suggest that the latex agglutination test (LA) could have sensitivity and specificity greater than observed. In fact, some cases in which culture, rapid urease test and histology were negative, the LA test was positive. The search for anti-H. pylori antibodies in some of these "false- positive" cases had demonstrated the presence of specific serum antibodies.
According to the experience of the Applicant, the 1-2% of patients hosts the coccoid form of H. pylori in the stomach, which retains its antigenicity (and therefore can be highlighted by the LA test), but cannot be cultured, and is not detectable by histologic examination.
2. Latex sensitized with antiserum against total proteins CagA+ H. pylori adsorbed with a suspension of a CagA" H. pylori strain
Two latex preparations have been compared:
1 . Latex sensitized with recombinant anti-CagA antibodies;
2. Latex sensitized with antibodies against total proteins CagA+ H. pylori adsorbed with a suspension of a CagA" H. pylori strain (G21).
Preliminary tests of the type of those specified in the previous section showed that the latex No. 1 produces an agglutination, with CagA+ strains, not so evident and difficult to be distinguished from that obtained with a CagA" (negative) strain.
A serum obtained as in the case No. 2 contains a much greater concentration IgGs against virulence antigens, compared to serum No. 1 , which was obtained by immunizing rabbits with the recombinant CagA protein only. Actually, as already noted, the wild CagA+ strains of possess an insertion, called "pathogenicity island"; that contains about thirty genes involved in virulence, and cagA is only one of these. These genes encode peptides with many different functions, which, overall, are used by the bacterium to move the CagA protein in the colonized gastric epithelial cell. These peptides are all immunodominant determinants and stimulate, in the infected individual and in immunized rabbits, a strong production of antibodies. In conclusion, a serum anti-total proteins of a CagA+ strain, adsorbed with a suspension of a CagA" strain, will contain many more antibodies, all directed against antigens involved in virulence.
The comparison between the performances of the two different prepa- rations showed the greater suitability of the preparation No. 2, as is illustrated in the two following tables.
Table 3 - Latex sensitized with anti-CagA antibodies
Table 4 - Latex sensitized with antibodies anti-total proteins of a
CagA+ strain adsorbed with a CagA" strain
3. Sensitization of a latex suspension with a rabbit serum consisting of antibodies against the antigens encoded by the cag genes
In order to identify those patients infected with H. pylori strains with determinants involving the highest risk of serious damage associated with infec- tion by Helicobacter, a latex coated with antibodies against all of the peptides encoded by cag genes, and not only by the cagA gene, was prepared.
In order to immunize rabbits the strain G33 (CagA+ and VacA+) described in the publication by Xiang et al. was used (Z. Xiang et al., Analysis of Expression of CagA and VacA Virulence Factors in 43 Strains of Helicobacter pylori Reveals that Clinical Isolates Can Be Divided into Two Major Types and that CagA is not Necessary for Expression of the Vacuolating Cytotoxin, Infect Immun Jan. 1995, 63: 94-98), as isolated from a patient with chronic gastritis of high degree, fairly active, with severe atrophy and moderate intestinal metaplasia of fundus. This pathological condition exposes the patient to a higher risk of developing gastric cancer. The characteristics of the strain are reported
in the cited publication.
The animal was immunized intravenously (through the marginal ear vein) with seven doses of a suspension of strain G33, each consisting of approximately 1010 microorganisms per ml in PBS with 0.5% formalin. The im- munization was carried out every 3-4 days. After the V dose, small bleedings were performed and the rabbit was finally deprived of all the blood that was possible to obtain (about 50 ml), and then the rabbit was sacrificed when the level of serum agglutinating antibodies had reached the value 1 :2000. After sensitizing latex with the serum obtained, and after testing the sensitivity and specificity thereof, the anti-G33 serum was adsorbed with the H. pylori G21 strain, non cytotoxic and free of the cag island (Xiang et al., ibidem).
The adsorption was carried out in the following way: G21 was inoculated on ten agar-blood plates, which were incubated in a microaerobic atmosphere at 37°C for 72 hours. The bacterial film was then collected and washed twice with PBS; 1 g of wet paste was then mixed thoroughly with 0.5 ml of anti-G33 serum (final dilution 1 :4 in PBS). The mixture was placed to incubate in normal air at 37°C for two hours, with frequent agitation in Vortex; subsequently, it was placed to incubate at 4°C overnight (in order to allow adsorption of incomplete antibodies).
Finally, the mixture was centrifuged at 4000 g at 4°C for 20 min and the adsorbed serum was collected and used for sensitizing a latex suspension. The latex so sensitized was then used to identify cag positive strains cultures on plate and in gastric biopsies.
In Figure 2 of the accompanying drawings agglutination obtained with latex sensitized with anti-CagA (upper left) and with anti-bodies anti-Cag antigens (top right and bottom left; at bottom right negative control consisting of a cag-negative strain) are observed.
In Figure 3 a positive result may be seen, to the left (agglutination of latex sensitized with Cag anti-antigens with two gastric biopsies from the same patient) and a negative case, to the right (gastric mucus obtained from a non-infected patient.
The present invention has been disclosed with particular reference to
some embodiments thereof, but it should be understood that modifications and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims
Claims
1. A method for rapid in vitro diagnosis of infection by highly pathogenic strains of Helicobacter pylori comprising the following steps:
a) contacting a suspension of staphylococci S. aureus Cowan 1 , or a suspension of latex particles, sensitized with anti-CagA antiserum, or with an antiserum against total proteins of CagA-positive H. pylori from which the other antibodies against H. pylori antigens have been separated, with a biological sample to be analyzed for the presence of the CagA protein antigen, or of pro- tein antigens encoded by the cag genes of H. pylori, and mixing said suspension with said biological sample;
b) visually detecting the possible agglutination of the mixture resulting from the previous step;
wherein the agglutination of said mixture shows the presence, in the biological sample under examination, of highly pathogenic Helicobacter pylori strains, which express the CagA protein or more proteins encoded by the cag genes.
2. A diagnostic method according to claim 1 , wherein said biological sample consists in gastric mucus, stools or in a biopsy specimen of the gastric mucosa,
3. A diagnostic method according to claims 1 o 2, wherein said step a) consists of contacting a suspension of latex particles sensitized with an antiserum against total proteins of CagA-positive H. pylori and subsequently adsorbed with a strain of CagA-negative H. pylori with a biological sample to be analyzed for the presence of the CagA protein antigen or of protein antigens encoded by cag genes, and mixing said suspension with said biological sample.
4. A diagnostic method according to claim 3, wherein said visual detection b) is carried out with the aid of a positive control sample consisting of a suspension of CagA-positive H. pylori, which is subjected, as the biological sample to be analyzed, to operations corresponding to steps a) and b).
5. A diagnostic method according to claim 4, wherein said visual detection b) is carried out with the aid of a negative control sample consisting of a
suspension of staphylococci or latex particles sensitized or not sensitized with hyperimmune serum, which is used in place of the suspension of step a) when subjecting the biological sample to be analyzed to operations corresponding to the steps a) and b).
6. A diagnostic method according to claims 3 or 4, further comprising the following steps:
c) contacting a suspension of staphylococci S. aureus Cowan 1 , or a suspension of latex particles, sensitized with antiserum against total H. pylori antigens, with a biological sample to be analysed for the presence of H. pylori antigens, and mixing said suspension with said biological sample; and
d) visually detecting the possible agglutination of the mixture resulting from the previous step;
wherein the agglutination of said mixture shows the presence, in the biological sample under examination, of Helicobacter pylori strains, and
the combined performance of the mentioned operations a), b, c) and d) allows detecting both infection by H. pylori and the presence of highly pathogenic Helicobacter pylori strains.
7. A diagnostic kit for rapid in vitro diagnosis of infection by highly pathogenic strains of Helicobacter pylori comprising the following components:
A. a suspension of staphylococci S. aureus Cowan 1 , or a suspension of latex particles, sensitized with anti-CagA antiserum, or with an antiserum against CagA-positive H. pylori from which the other antibodies against H. pylori antigens have been separated, in an aqueous solution such as phosphate-buffered saline (PBS) or PBS-formalin, containing an antimicrobial agent, such as merthiolate or sodium azide;
B. an aqueous solution such as PBS or PBS-formalin, containing a mucus-fluidifying agent, such as dithiothreitol, and an antimicrobial agent, such as merthiolate or sodium azide;
C. some cardboard sheets with dark disks, on which the test is to be performed.
8. A diagnostic kit according to claim 7, wherein said component A consists of a suspension of latex particles sensitized with an antiserum against to-
tal proteins of CagA-positive H. pylori and subsequently adsorbed with a strain of CagA-negative H. pylori in an aqueous solution as phosphate buffered saline (PBS) or PBS-formalin, containing an antimicrobial agent, as merthiolate or sodium azide.
9. A diagnostic kit according to claim 8, which includes, in addition:
D1 . a suspension of CagA-positive H. pylori antigens, in an aqueous solution such as PBS or PBS-formalin, and an antimicrobial agent, such as merthiolate or sodium azide, as positive control for CagA-positive H. pylori.
10. A diagnostic kit according to claims 8 or 9, which includes, in addition:
D2. a suspension of H. pylori antigens, in an aqueous solution such as PBS or PBS-formalin, and an antimicrobial agent, such as merthiolate or sodium azide as a positive control for H. pylori.
1 1 A diagnostic kit according to any one of claims 7-10, wherein said component B contains dithiothreitol in a concentration comprised between 0.05 and 1 % by weight.
12. A diagnostic kit according to claim 9, wherein said aqueous solution is a phosphate buffered saline (PBS) or a phosphate buffered saline contain- ing 0.5-1 .0% by weight of formalin (PBS-formalin), and said antimicrobial agent is merthiolate in a concentration of about 0.05% by weight or sodium azide in a concentration of about 0.1 % by weight.
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CN116375823A (en) * | 2022-12-09 | 2023-07-04 | 扬州大学 | B cell epitope target antigen carrying helicobacter pylori virulence factor, expression vector and application thereof |
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CN113281509A (en) * | 2021-05-29 | 2021-08-20 | 北京华宇亿康生物工程技术有限公司 | Preparation method of kit for detecting helicobacter pylori antigen with good stability |
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